Electron spin resonance spectra recorded from 5-nitroxide stearate (5-NS) labeled rat adipocyte ghosts incubated with various concentrations of CaCl2. Ca2 ion (0.5 to 3.0 mM) decreased the lipid fluidity, as indicated by increases in the order parameter (S); 2.6 mM Ca2 ion elevated S by 3% at 37 degrees C. This effect may be mediated by Ca2 ion binding to low affinity sites on the plasma membrane and is comparable to reducing the temperature by 3 degrees C. Ca2 ion may play central roles in the action of insulin on fat cell function, and it is possible that appropriately rigid membrane lipids are involved. The viability and hormonal responsiveness of intact adipocytes were assessed by measuring the uptake of glucose from the incubation medium in the presence and absence of insulin. The addition of insulin (at a concentration that stimulates glucose uptake in the intact cells) to 5-NS labeled fat cell ghosts (with or without Ca2 ion) caused no detectable change in the fluidity. A detailed study was also conducted on the in vitro effects of clinical grade human growth hormone (hGH), intact monomeric hGH, and a naturally-occurring diabetogenic substance purified from clinical grade hGH on the fluidity of rat adipocyte ghosts and human erythrocyte ghosts. No effects of these agents were noted on the fluidity of the 5-NS labeled membranes under a variety of incubation conditions. The effect of temperature on the spectra of the 5-NS labeled fat cell ghosts was studied from 10 to 40 degrees C. A lipid phase separation was detected, having an onset temperature of approximately 30 degrees C. It is likely that at temperatures below 30 degrees, "clusters" of lipid form which exclude the incorporated spin label and also alter the fluidity of the lipid sampled by the probe. Lipid structrual changes induced by the phase separation might partially mediate the effects which temperature exerts on certain fat cell membrane functions, such as glucose transport, and the binding, degradation and action of insulin.